Detecting succinylacetone

ABSTRACT

This invention relates, inter alia, to detecting and/or measuring succinylacetone and one or more additional biological analytes using mass spectrometry.

CLAIM OF PRIORITY

This application is a continuation and claims the benefit of priorityunder 35 U.S.C. §120 of U.S. application Ser. No. 13/092,584, filed onApr. 22, 2011, which is a continuation of U.S. application Ser. No.11/744,789, filed on May 4, 2007, now U.S. Pat. No. 7,951,608, theentire contents of which are incorporated herein by reference.

BACKGROUND

Mass spectrometry is useful for detecting and measuring a wide varietyof metabolites, the presence or amount of which can be indicative ofcertain conditions or disorders. Thus, mass spectrometry can be used,e.g., to diagnose numerous metabolic disorders associated with alteredlevels of metabolites. One such metabolic disorder is hereditarytyrosinemia, Type I (HT1), which is caused by a deficiency offumarylacetoacetate hydrolase (FAH) and is associated with increasedlevels of tyrosine and succinylacetone. HT1 is a childhood disorder thatcauses liver failure, painful neurological crises, rickets, andhepatocarcinoma. If untreated, death typically occurs at less than 2years of age, with some chronic forms allowing survival to 12 years ofage. It is now possible to treat HT1 with NTBC (or Nitisinone), iftreatment is initiated early in life. Thus, there is a major incentiveto identify HT1 affected patients by newborn screening or even prenatalscreening.

SUMMARY

Succinylacetone is the primary marker for the early detection of HT1.However, succinylacetone is a very reactive ketone that forms conjugateswith proteins. The methods described herein can be used to extractsuccinylacetone from a sample under conditions that permit concurrentlyextracting other metabolites, such as amino acids, free carnitine, oracylcarnitines. For example, harsh extractions conditions (such asextreme acidity and high temperature) can be avoided.

The methods described herein can be used to detect and/or measuresuccinylacetone and one or more additional biological analytes usingmass spectrometry (e.g., tandem mass spectrometry). Such methods areuseful in diagnosis and for generating metabolic profiles for thedetection/diagnosis of metabolic disorders such as amino acidopathies(e.g., tyrosinemia type I).

In one aspect, the disclosure provides a method for extraction. Themethod includes the steps of: contacting a sample with an extractionsolution, the extraction solution comprising a C1-3 linear or branchedchain monoalcohol and a strong base, wherein contacting the sample withthe extraction solution yields an extract comprising: (i) derivatizedsuccinylacetone, (ii) one or more amino acids, (iii) free carnitine,(iv) one or more acylcarnitines, or (iv) derivatized forms of any of(ii), (iii), or (iv) from the sample, and wherein the concentration ofthe derivatized succinylacetone in the extract reflects theconcentration of succinylacetone in the sample, and wherein theconcentrations of the one or more amino acids, free carnitine, one ormore acylcarnitines, or derivatized forms thereof in the extract reflecttheir respective concentrations in the sample. The method can alsoinclude the step of after contacting the sample with the extractionsolution, analyzing the sample using tandem mass spectrometry. Thecontacting can derivatize at least one succinylacetone molecule to3-(5-methyl-1H-pyrazol-3-yl) propionic acid (MPP). The C1-3 linear orbranched chain monoalcohol can be methanol, ethanol, propanol, orisopropanol. The strong base can be hydrazine, a modified hydrazine(e.g., acyl-hydrazines, aryl-hydrazines, alkyl-hydrazines, Girard-P andGirard-T reagents), or hydroxylamine.

In some embodiments, the sample can be a biological sample such as adried blood spot. The sample can be one obtained from a newborn human.The sample can be one obtained from a subject suspected of, or a risk ofdeveloping, a metabolic disorder such as tyrosinemia type I.

In some embodiments, the extraction solution can contain water, anorganic acid, and/or one or more internal standards. For example, theextraction solution can contain between about 5% to about 30% water orbetween about 20% to about 25% water. The organic acid can be oxalicacid, e.g., at a concentration of about 5 mM. The internal standards caninclude at least one heavy atom isotope such as ²H, ¹⁵N, or ¹³C. One ormore of the internal standards can be, or contain, succinylacetone, anamino acid, free carnitine, an acylcarnitine, or derivatized form of anyof the aforementioned.

In some embodiments, the method can include the steps of aftercontacting the sample with the extraction solution, evaporating thesample resulting in a first evaporated sample. The method can furtherinclude the steps after evaporating the sample, contacting the firstevaporated sample with an alkyl alcohol solution comprising an alkylalcohol and an acid. The method can further include the steps of: aftercontacting the sample with the alkyl alcohol solution, evaporating thesolution resulting in a second evaporated sample and/or reconstitutingthe second evaporated sample. Reconstituting the second evaporatedsample can include contacting the second evaporated sample with asolvent. The alkyl alcohol can be methanol, ethanol, propanol,isopropanol, n-butanol, t-butanol, or pentanol. The acid can behydrochloric acid (HCl). The solvent can include acetonitrile,isopropanol, or a mixture of water with isopropanol or acetonitrile.

In another aspect, the disclosure provides a method for extraction,which method includes the steps of: contacting a sample with anextraction solution, the extraction solution comprising a C1-3 linear orbranched chain monoalcohol and a strong base, wherein contacting thesample with the extraction solution yields an extract comprising (i) aderivatized biologically active ketone, (ii) one or more amino acids,(iii) free carnitine, (iv) one or more acylcarnitines, and/or (v)derivatized forms of any of (ii), (iii), or (iv) from the sample, andwherein the concentration of the derivatized biologically active ketonein the extract reflects the concentration of the biologically activeketone in the sample, and wherein the concentrations of the one or moreamino acids, free carnitine, one or more acylcarnitines, or derivatizedforms thereof in the extract reflect their respective concentrations inthe sample. The C1-3 linear or branched chain monoalcohol can bemethanol, ethanol, propanol, or isopropanol. The biologically activeketone can be, e.g., succinylacetone or a steroid. Steroids include, butare not limited to, testosterone dehydroepiandrosterone (DHEA),dehydroepiandrosterone sulfate, adrostenedione, 17-hydroxyprogesterone(17-OHP), 17-hydroxy pregnenolone, cortisol, 11-deoxycortisol,corticosterone, aldosterone, estradiol, 18-OH corticosterone,pregnenolone, progesterone, cortisone, terta-hydrocortisol,11-deoxycorticosterone, creatinine, 17-Ketosteroids, cholesterol,vitamin B, or vitamin A. The strong base can be any of the strong basesdescribed herein. The extraction solution can also include water.

In another aspect, the disclosure features a method for detectingsuccinylacetone. The method includes the steps of: contacting a samplewith an extraction solution comprising a C1-3 linear or branchedmonoalcohol and a strong base; derivatizing succinylacetone in thesample; and evaluating the derivatized succinylacetone in thederivatized sample using tandem mass spectrometry. The derivatized formof succinylacetone can be succinylacetone to3-(5-methyl-1H-pyrazol-3-yl) propionic acid (MPP). The strong base canbe any described herein.

In another aspect, the disclosure provides a method for detectingsuccinylacetone, which method includes the steps of contacting a samplewith an extraction solution comprising a C1-3 linear or branchedmonoalcohol and hydrazine; derivatizing succinylacetone to3-(5-methyl-1H-pyrazol-3-yl) propionic acid (MPP) in the sample; andevaluating MPP in the derivatized sample using tandem mass spectrometry.The method can also include evaluating the sample for one or moreadditional analytes (e.g., any of the additional analytes describedherein) with MPP in the same sample injection. The C1-3 linear orbranched chain monoalcohol can be methanol, ethanol, propanol, orisopropanol.

In another aspect, the disclosure provides a method for detectingsuccinylacetone. The method can include the steps of: contacting asample with an extraction solution containing an organic solvent underconditions that do not substantially fix proteins; derivatizingsuccinylacetone to 3-(5-methyl-1H-pyrazol-3-yl) propionic acid (MPP) inthe sample; and evaluating MPP and an additional analyte (or derivativethereof) in the derivatized sample using tandem mass spectrometry. Theextraction solution can contain about 5% water. The extraction solutioncan contain about 85% of a C1-3 linear or branched monoalcohol such asmethanol, ethanol, propanol, or isopropanol. Succinylacetone can bederivatized with hydrazine or derivatized hydrazine.

In some embodiments, the method can include the steps of determiningwhether a subject, from whom the sample was derived, has, or is at riskof developing, hereditary tyrosinemia type I, based on the detection ofsuccinylacetone in the sample. After determining that a subject has, oris at risk of developing, hereditary tyrosinemia type I, the method caninclude administering to the subject an inhibitor of4-hydroxyphenylpyruvate dioxygenase.

In yet another aspect, the disclosure features a method for detecting abiologically active ketone. The method can include the steps of:contacting a sample with an extraction solution comprising a C1-3 linearor branched monoalcohol and a strong base; derivatizing a biologicallyactive ketone in the sample; and evaluating the derivatized biologicallyactive ketone in the derivatized sample using tandem mass spectrometry.The biologically active ketone can be succinylacetone or a steroid suchas any of the steroids described herein. The C1-3 linear or branchedchain monoalcohol can be methanol, ethanol, propanol, or isopropanol.The extraction solution can also include water.

In some embodiments of any of the methods described herein, the samplecan be a biological sample such as a dried blood sample (a dried bloodspot). The sample can be from a newborn human. The sample can alsoinclude at least one heavy atom isotope is included in the sample priorto mass spectroscopic analysis.

In some embodiments of any of the methods described herein, theevaluating can include analyzing derivatized succinylacetone along withone or more additional analytes (e.g., biological analytes such as oneor more amino acids, free carnitine, or acylcarnitines) from the sampleinjection into a tandem mass spectrometer. For example, derivatizedsuccinylacetone can be analyzed along with one or more (e.g., two,three, four, five, six, seven, eight, nine, 10, 11, 12, 15, 18, 20, 22,25, 28, or 30) additional analytes from the same sample injection (intoa tandem mass spectrometer).

In some embodiments of any of the methods described herein, the sampleis one that has not been previously extracted.

In some embodiments of any of the methods described herein, contactingthe sample with the extraction solution results in the extraction of (i)derivatized succinylacetone (SA) and (ii) one or more amino acids, freecarnitine, one or more acylcarnitines, or derivatized forms thereof fromthe sample without altering the ratios of these analytes present in thesample. For example, an extract obtained from a sample containing SA,tyrosine, and free carnitine at a ratio of approximately 5:1:2 wouldcontain derivatized succinylacetone, tyrosine (or derivatized tyrosine),and free carnitine (or derivatized free carnitine) at a ratio ofapproximately 5:1:2.

In yet another aspect, the disclosure features a kit for detectingsuccinylacetone. The kit can include derivatized succinylacetonecomprising at least one heavy atom isotope; and instructions for how todetect the derivatized succinylacetone. The kit can also include astrong base such as hydrazine. The strong base can be provided at aconcentration of less than about 0.1%. The hydrazine can be hydrazinedihydrochloride. The kit can also include one or more internalstandards, each internal standard containing: (i) an amino acid, freecarnitine, or an acylcarnitine and (i) at least one heavy atom isotope.The kit can also include at least one dried blood spot comprising aknown amount of one or more of succinylacetone, an amino acid, freecarnitine, or an acylcarnitine. The derivatized succinylacetone can be3-(5-methyl-1H-pyrazol-3-yl) propionic acid (MPP).

In another aspect, the disclosure provides a kit for detecting abiologically active ketone. The kit can include a derivatizedbiologically active ketone of interest containing at least one heavyisotope atom and instructions for how to detect the derivatizedbiologically active ketone.

Many mass spectrometers have mass accuracies to high resolution. Forexample, in the case of a singly charged ion, this range corresponds to0.6 m/z. Minor variations (e.g., variations in the calibration) in amass spectrometer may result in ion m/z signals that do not coincidewith the ones stated herein, but the m/z signal corresponding to thosedisclosed can be easily identified and used, e.g., by compensating foroffset in calibration.

All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram depicting the pathway of tyrosinecatabolism.

FIG. 2 is a schematic diagram depicting a reaction necessary to extractand derivatize succinylacetone from a biological sample.

FIG. 3 is a schematic diagram of a method of extracting and derivatizingsuccinylacetone prior to injection and detection/measurement by tandemmass spectrometry.

FIG. 4 is a schematic diagram of a method of extracting and derivatizingsuccinylacetone, and extracting and esterifying additional analytesprior to injection and detection/measurement by tandem massspectrometry.

FIG. 5 is a tandem mass spectrum (Neutral loss of 46 scan) acquiredusing the method depicted in FIG. 3 of derivatized succinylacetone andother non-derivatized amino acids. Amino acids are designated bythree-letter code, e.g., alanine is “ala,” and the specificmass-to-charge ratio (m/z) signal for a daughter ion of an individualamino acid is indicated by arrow. “IS” refers to internal standard. TheX-axis represents the m/z and the Y-axis represents the relativeabundance (percentage) of each ion in the sample.

FIG. 6 is a tandem mass spectrum (Neutral loss of 102 scan) of anextracted blood spot depicting exemplary analytes that can be detectedand/or measured together with succinylacetone when the sample isprocessed according to the method depicted in FIG. 4.

FIG. 7 is a series of spectra (Neutral loss of 46 scan) acquired withthe method described in FIG. 3. The spectra depict the measurement ofsuccinylacetone and tyrosine levels in dried blood spots from healthynewborns and from a newborn confirmed as having tyrosinemia type I. TheX-axis represents the corresponding m/z and the peaks heights representthe relative abundance of the analytes.

FIG. 8 are a pair of bar graphs and a table depicting the measurement ofsuccinylacetone and tyrosine levels in dried blood spots obtained from anewborn confirmed as having tyrosinemia type I at 25 hours and 15 days,as compared to a healthy newborn (“true neg”). The Y-axis of the bargraph of FIG. 8A represents the level of succinylacetone in μM (left).The Y-axis of the bar graph of FIG. 8B represents the level of tyrosinein μM (right).

FIG. 9 is a line graph depicting the relative stability of hydrazinehydrate and hydrazine dihydrochloride. The X-axis represents the numberof days and the Y-axis represents the amount (percentage) of hydrazinehydrate (diamonds) or hydrazine dihydrochloride (squares) remaining ateach time point.

DETAILED DESCRIPTION

Succinylacetone can be detected by mass spectrometry by modifyingsuccinylacetone in a sample to a more stable form. Disclosed herein aremethods and compositions for performing such modifications and forconcurrently treating the sample in a manner that allows for theextraction of succinylacetone and other analytes (e.g., amino acids,acylcarnitines, and free carnitine) from a sample in a single step suchthat the concentrations of succinylacetone and the other analytes in theextract reflect their respective concentrations in the sample. Alsodisclosed are methods for detecting and/or measuring succinylacetone(derivatized succinylacetone) and one or more additional analytes usingmass spectrometry. The methods described herein can be used, inter alia,for diagnosing one or more metabolic disorders in a subject such asamino acidopathies (e.g., Hereditary tyrosinemia type I) and disordersof organic and fatty acid metabolism or for generating metabolicprofiles for such diagnoses (see below).

Methods for Extracting Succinylacetone and Additional Analytes from aSample

The disclosure features methods for extracting succinylacetone alongwith one or more additional analytes (e.g., amino acids, acylcarnitines,and free carnitine) from the sample in a single step such that theconcentrations of succinylacetone and one or more additional analytes(e.g., amino acids, free carnitine, and acylcarnitines) in the extractreflect their respective concentrations in the sample. Following theextraction, the presence or amount of succinylacetone can be determinedalong with one or more additional analytes (e.g., free carnitine,acylcarnitines, and amino acids) using mass spectrometry (e.g., tandemmass spectrometry). The methods can include contacting a sample with anextraction solution containing a C1-3 linear or branched chainmonoalcohol (e.g., methanol, ethanol, propanol, or isopropanol) and astrong base such as hydrazine, a modified hydrazine (e.g.,acyl-hydrazines, aryl-hydrazines, alkyl-hydrazines, Girard-P andGirard-T reagents), or hydroxylamine. The extraction solution can alsocontain water. Contacting the sample with the extraction solutionresults in modification (derivatization) of succinylacetone, if presentin the sample and the extraction of the modified succinylacetone as wellas one or more additional analytes (e.g., free carnitine,acylcarnitines, and amino acids).

FIG. 2 depicts an exemplary reaction for extracting succinylacetone fromthe dried blood spot samples according to the methods described herein.Succinylacetone is a very reactive diketone and thus it reacts rapidlywith the side chains of certain amino acids (e.g., free amino acids orconstituents of peptides and proteins) in biological fluids such aswhole blood. The Schiff base conjugates formed by the reaction betweensuccinylacetone and the amino acid residues are more stable than thefree succinylacetone and thus most, if not all, succinylacetone presentin blood is in the bound form. To extract (release) succinylacetonealong with one or more additional analytes from a sample (e.g., abiological sample such as a blood spot) in a single step, the sample canbe contacted with an extraction solution containing a C1-3 linear orbranched chain monoalcohol (e.g., methanol, ethanol, propanol, orisopropanol) and a strong base. The strong base can be hydrazine or amodified hydrazine (e.g., acyl-hydrazines, aryl-hydrazines,alkyl-hydrazines, Girard-P and Girard-T reagents) as well ashydroxylamine. The C1-3 linear or branched chain monoalcohol can be, forexample, at a concentration of about 70% (e.g., about 70%, about 71%,about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%,about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about91%, about 92%, about 93%, about 94%, or about 95%) by volume in thesolution. The strong base (e.g., hydrazine, modified hydrazine, orhydroxylamine) can be at a concentration of about 600 μM (e.g., about200 μM, about 300 μM, about 400 μM, about 500 μM, about 550 μM, about650 μM, about 700 μM, about 800 μM, about 900 μM, about 1,000 μM, about1,200 μM, about 1,500 μM, or about 2,000 μM) in the solution.

The extraction solution can also contain water. The water can be, forexample, at a concentration of 6-30% (e.g., 7-28%, 8-26%, 10-26%,14-25%, 18-24%) by volume in the extraction solution. The concentrationof water can be such that the extraction solution reconstitutes some ofthe proteins and peptides while at the same time dissolving otheranalytes (e.g., acylcarnitines, free carnitine, and amino acids) presentin the sample. The extraction solution can also contain an organic acidsuch as oxalic acid at a concentration of about 3 mM (e.g., about 1 mM,about 2 mM, about 2.5 mM, about 3.5 mM, about 4 mM, about 4.5 mM, orabout 5 mM).

Optionally, with the aid of the organic acid (e.g., oxalic acid), whichacts as a catalyst, hydrazine releases succinylacetone from the aminoacid residues and forms a stable pyrazone ring. The pyrazone formed bythe reaction between succinylacetone and hydrazine is the compoundcalled 3-(5-methyl-1H-pyrazol-3-yl)propionic acid (MPP). This compoundis more stable than the Schiff bases form by the reaction ofsuccinylacetone with amino acid residues. Therefore, oncesuccinylacetone has reacted with hydrazine, this MPP derivative can befully extracted along with additional analytes (e.g., amino acids, freecarnitine, and acylcarnitines) in a single step. The extracted MPP andadditional analytes can then be measured using tandem mass spectrometry.Succinylacetone and other analytes can be evaluated, for example,concurrently using tandem mass spectrometry. The concentration of MPPdirectly reflects the concentration of succinylacetone in the sample.

FIG. 3 depicts an exemplary method for preparing a sample for massspectrometric analysis. In this case, a dried blood spot sample obtainedfrom a newborn (or person of any age) can be perforated to generate asmall disc that is deposited, e.g., in a well of a microtiter plate. Tothis sample, an extraction solution can be added to extract the analytesin the sample. The extraction solution can comprise a mixture of a C1-3linear or branched chain monoalcohol and a strong base (the proportionsof these two components can vary as described above). The source ofhydrazine can be hydrazine hydrate or hydrazine dihydrochloride, orother modified hydrazines (e.g., acyl-hydrazines, aryl-hydrazines,alkyl-hydrazines, Girard-P and Girard-T reagents) or hydroxylamine Thesolution can also contain water (see above) with a small percentage ofan organic acid (e.g., oxalic acid). This solution can also, optionally,contain one or more internal standards for, e.g., amino acids, freecarnitine, acylcarnitines and succinylacetone at known concentrations.The sample mixture can then be incubated for a pre-determined period oftime (e.g., about 25 to about 45 minutes (e.g., about 30 to about 45minutes; about 30 to about 60 minutes; about 30 to about 70 minutes;about 30 to about 90 minutes; about 30 to about 120 minutes; about 35 toabout 60 minutes; or about 40 to about 60 minutes) to allow theextraction of amino acids, free carnitine and acylcarnitines as well asthe extraction of bound succinylacetone and its concomitant reactionwith hydrazine to occur. The extract can then be transferred to anunused well of a micro titer plate and the samples then analyzed bytandem mass spectrometry, optionally, with the aid of a liquid handlingdevice for sample injection. The instrumental settings on the tandemmass spectrometer are then set to detect the respective metabolites ofinterest (amino acids, acylcarnitines, carnitine, and succinylacetone)as well as their corresponding internal standards in a multiplexfashion.

It can be advantageous to derivatize not only the modifiedsuccinylacetone (e.g., MPP), but additional analytes in a sample (e.g.,additional analytes such as amino acids, acylcarnitines, and carnitine).Many of the analytes described herein, including succinylacetone, arecarboxylic acids; therefore, they are amenable for sample derivatizationby esterification. An exemplary method for esterifying multiple analytesin a sample, prior to analysis by mass spectrometry, is depicted in FIG.4. First, a similar procedure can be performed as above, however,further sample processing can be performed. For example, following thederivatization step with hydrazine, the sample can be evaporated todryness. The dried sample can then be reconstituted in an acidicsolution of an alkyl alcohol. The alcohol can be any alkyl alcohol suchas, but not limited to, methanol, ethanol, propanol, n-butanol,tert-butanol, pentanol, or hexanol. This alcohol can be contacted withthe sample in combination with a strong, concentrated acid (e.g.,hydrochloric acid or sulfuric acid). Such a solution of an alkyl alcoholand an acid can be, for example, butanol in 3N HCl or methanol in 1NHCl. The sample can be incubated in the alkyl alcohol/acid solution forabout 30 minutes (e.g., about 20 minutes, about 40 minutes, about 45minutes, about 50 minutes, about 60 minutes, about 70 minutes, about 80minutes, about 90 minutes, about 100 minutes, or about 120 minutes) atabout 39° C. (e.g., about 30° C., about 35° C., about 36° C., about 37°C., about 40° C., about 42° C., about 50° C., about 55° C., about 60°C., or about 70° C.). Following this incubation, the sample can beevaporated to dryness and then reconstituted in a solvent (e.g.,acetonitrile; or acetonitrile and water (e.g., 80% acetonitrile and 20%water), isopropanol (e.g., 80% isopropanol and 20% water) or any othersolvent that is amenable for mass spectrometry analysis and that iscapable of dissolving esterified organic compounds.

Additional analytes that can be detected and/or measured withderivatized (modified) succinylacetone include, e.g., those listed inTable 1.

TABLE 1 ANALYTE NAME ABBREVIATION Ketones Succinylacetone SA Amino acidsAlanine Ala Arginine Arg Aspartic Acid Asp Asparagine Asn Citrulline CitCysteine Cys Glycine Gly Glutamine Gln Glutamic Acid Glu Histidine HisLeucine (isoleucine, Allo-Isoleucine) Leu (Ile, Allo-Ile) Lysine LysMethionine Met Ornithine Orn Phenylalanine Phe Proline Pro Serine SerThreonine Thr Tryptophan Trp Tyrosine Tyr Valine Val Carnitines Freecarnitine C0 Acetylcarnitine C2 Propionylcarnitine C3 MalonylcarnitineC3DC Butyrylcarnitine C4 3-Hydroxy-butyrylcarnitine C4OHIsovalerylcarnitine C5 Tiglylcarnitine C5:1 Glutarylcarnitine C5DC3-Hydroxy-isovalerylcarnitine C5OH Hexanoylcernitine C6 AdipylcarnitineC6DC Octanoylcernitine C8 Octenoylcarnitine C8:1 Decanoylcarnitine C10Decenoylcarnitine C10:1 Decadienoylcarnitine C10:2 DodecanoylcarnitineC12 Dodecenoylcarnitine C12:1 Tetradecanoylcarnitine(Myristoylcarnitine) C14 Tetradecenoylcarnitine C14:1Tetradecadienoylcarnitine C14:2 3-Hydroxy-tetradecanoylcarnitine C14OHHexadecanoylcarnitine (palmitoylcarnitine) C16 HexadecenoylcarnitineC16:1 3-Hydroxy-hexadecanoylcarnitine C16OH3-Hydroxy-hexadecenoylcarnitine C16:1OH Octadecanoylcarnitine(Stearoylcarnitine) C18 Octadecenoylcarnitine (Oleylcarnitine) C18:1Octadecadienoylcarnitine (Linoleylcarnitine) C18:23-Hydroxy-octadecanoylcarnitine C18OH 3-Hydroxy-octadecenoylcarnitineC18:1OH

Mass Spectrometry

Tandem mass spectrometry can be used to detect and/or measuresuccinylacetone and one or more additional analytes (e.g., freecarnitine, acylcarnitines, and amino acids) in a sample (e.g., abiological sample). In tandem mass spectrometry, two mass analyzers arelinked in series via a collision cell. The first mass analyzer (MS-1) isused to select an ion of interest (e.g., an ion of a particularmass-to-charge ratio (m/z)). The selected ions are then transferred to acollision cell where they are fragmented by collisions with an inertgas. This process is called collisionally activated dissociation (CAD).Once the parent (sometimes referred to as precursor) ions havefragmented, the second mass analyzer (MS-2) is used to either scan anddetect all of the produced daughter ions or to select and detectparticular fragment ions.

As detailed in the accompanying Examples, tandem mass spectrometry wasused to ionize the precursor molecules of derivatized (modified)succinylacetone and several amino acids, fragment the ions, and detectspecific peaks that are indicative of the presence of these molecules inthe sample. The tandem mass spectrometry detection can be accomplishedin a number of ways. In one type of tandem mass spectrometry (commonlyperformed on triple quadrupole tandem mass spectrometers) ions thatfragment to produce common daughter (fragment) ions can be detected as aclass by performing a “precursor ion scan” (also called parent ionscan), where by selecting the appropriate mass for the common fragmention in MS-2 all ion that produce the common fragment ions are detected.This type of scan can be used to detect the acylcarnitines in a sample(precursor ion of m/z 85 scan). In a different form of tandem massspectrometry, ions that fragment to produce a common neutral loss can bedetected as a class by performing a so called neutral loss scan where bysetting an appropriate mass offset equal to the common neutral lossbetween MS-1 and MS-2 all ions that fragment to produce the specifiedneutral loss are detected. This type of scan is performed to detectamino acids and succinylacetone in a sample (neutral loss of m/z 102 ifthe analytes in the extracted sample were modified in to butyl esters orneutral loss of m/z 46 if no further analyte modification occurred).FIG. 5 shows a neutral loss scan of m/z 46 were several amino acids andsuccinylacetone are detected from the same sample. A unique peakcorresponding to derivatized succinylacetone is observed at m/z 155,together with several unique peaks corresponding to amino acids. Thus,succinylacetone (derivatized succinylacetone as described herein) can bedetected and/or measured along with one or more additional analytes in asingle sample in one analysis.

In yet another type of tandem mass spectrometry known as multiplereaction monitoring (MRM), a parent ion of interest is selected in MS-1,fragmented in the collision cell and a specific fragment ion resultingfrom the collisional activation is selected in MS-2 and finallydetected. MS-1 and MS-2 are fixed to respectively select thecorresponding parent and fragment ion pairs of interest for apredetermined amount of time (a few milliseconds). This specific parention-product ion transition can be considered as one detection channel.If additional analytes need to be detected, additional detectionchannels with specific mass transitions can be introduced in theexperiment. The data from all selected mass transitions (channels) canbe acquired sequentially to obtain the desired information. Thedetection and quantitation of succinylacetone (derivatizedsuccinylacetone in a sample prepared as described herein) in a mixturecan be obtained by employing the specific mass transition for each ofthese compounds as follows: for derivatized succinylacetone: MS-1 fixedto select and transmit the parent ion at m/z 155, MS-2 fixed to selectand transmit the specific product ion at m/z 109 (channel 1 or MRMtransition 1); and for an amino acid, such as tyrosine: MS-1 fixed toselect and transmit the parent ion at m/z 182, MS-2 fixed to select andtransmit the specific product ion at m/z 136 (channel 2 or MRMtransition 2). These two MRM transitions can be measured sequentiallyfrom the same sample for a predetermined amount of time to detect thepresence and/or concentration of a mixture of these compounds in suchsample.

Stable isotope-labeled internal standards for succinylacetone(derivatized succinylacetone) can be added to a sample, by whichquantitation of derivatized succinylacetone, and thus succinylacetoneitself, can be performed. Such labeling of derivatized succinylacetonewith stable isotopes results in a mass shift, while retaining verysimilar physicochemical properties between the labeled and unlabeledcompounds.

Generally, one or more internal standards can be added at knownconcentration to a sample to allow for quantitation of the analyte ofinterest (e.g., succinylacetone). For example, for a sample analyzedusing tandem mass spectrometry, the ratio of the signals produced byderivatized succinylacetone (e.g., MPP) and its corresponding internalstandard can be used to determine the amounts of this compound in thesample. The internal standard can also be added to distinguish naturallyoccurring (endogenous) molecules. As above, the internal standards canbe prepared in an extraction solution prior to mixing a sample (e.g., ablood sample) and the extraction solution. Alternatively, the internalstandards can be added to the mixture at any step in the samplepreparation that ensures these internal standards will not be removedfrom the mixture during the sample processing (e.g., after aliquid-liquid extraction or a solid phase extraction).

Internal standards for an analyte of interest (or other molecules, e.g.,biomolecules described herein) detected by a method described herein canbe any modification or analog of that analyte molecule that isdetectable by mass spectrometry. An internal standard is separatelydetectable from the molecule based on unique physical characteristics,such as a unique mass or mass-to-charge ratio. A commonly used internalstandard for mass spectrometry is a stable isotopically labeled form orchemical derivative of an analyte of interest (e.g., if the analyte wasMPP, the internal standard can be an isotopically labeled MPP). Forexample, stable isotope labeled analogs can be used to quantitate thecorresponding analyte of interest using the technique known as isotopedilution mass spectrometry where the analyte and internal standards areprocessed in the same sample. Internal standards can be designed suchthat 1) the labeling causes a shift in mass of at least 1 mass unit and2) that none of the stable isotope labels are located in labile sites toprevent exchange. Labels can be ²H (D), ¹⁵N, ¹³C or ¹⁸O in anycombination. The actual location of the labels on the molecule can varyprovided the pre-requisite 2 (above) is satisfied. Moreover, theposition of the labels and the potential change in the mass of thefragment ions can also be used to confirm separation of the internalstandard and analytes. Examples of potential internal standards usefulin the methods described herein include, but are not limited to, anisotopically labeled: derivatized succinylacetone (e.g.,3-(5-methyl-1H-pyrazol-3-yl)propionic acid (MPP)), carnitine,acylcarnitine, or amino acid (e.g., proline, methionine, or tyrosine).

Several types of mass spectrometers are available or can be producedwith various configurations, all of which can be useful in the methodsdescribed herein. In general, a mass spectrometer has the followingmajor components: a sample inlet, an ion source, a collision cell, amass analyzer, a detector, a vacuum system, and instrument-controlsystem, and a data system. Difference in the sample inlet, ion source,and mass analyzer generally define the type of instrument and itscapabilities. For example, an inlet can be a capillary-column liquidchromatography source or can be a direct probe or stage such as used inmatrix-assisted laser desorption. Common ion sources are, for example,electrospray, including nanospray and microspray or matrix-assistedlaser desorption. Common mass analyzers include quadrupole mass filters,time-of-flight mass analyzers (preferably an orthogonal accelerationtime-of-flight mass analyzer), ion trap mass filters, magnetic sectoranalysers, or Fourier Transform Ion Cyclotron Resonance (“FTICR”) massanalysers. The collision cell can be, e.g., a quadrupole rod set, ahexapole rod set, or an octopole rod set. The collision cell preferablyforms a substantially gas-tight enclosure apart from an ion entrance andion exit aperture. A collision gas such as helium, argon, nitrogen, airor methane may be introduced into the collision cell.

The specific examples described herein were performed using tandem massspectrometers (see, e.g., the accompanying Examples).

Samples

Suitable samples for the methods described herein include any biologicalfluid, cell, tissue, or fraction thereof, that includes biomoleculesindicative of a metabolic state (e.g., a metabolic disordercharacterized by altered succinylacetone levels such as Hereditarytyrosinemia type I). A sample can be, for example, a specimen obtainedfrom a subject (e.g., a mammal such as a human) or can be derived fromsuch a subject. For example, a sample can be a tissue section obtainedby biopsy, or cells that are placed in or adapted to tissue culture.Exemplary samples therefore include cultured fibroblasts, culturedamniotic fluid cells, and chorionic villus sample. A sample can also bea biological fluid specimen such as urine, blood, plasma, serum, saliva,semen, sputum, cerebral spinal fluid, tears, mucus, and the like. Asample can be further fractionated, if desired, to a fraction containingparticular cell types. For example, a blood sample can be fractionatedinto serum or into fractions containing particular types of blood cellssuch as red blood cells or white blood cells (leukocytes). If desired, asample can be a combination of samples from a subject such as acombination of a tissue and fluid sample, and the like. Methods forobtaining samples that preserve the activity or integrity of moleculesin the sample are well known to those skilled in the art. Such methodsinclude the use of appropriate buffers and/or inhibitors, includingnuclease, protease and phosphatase inhibitors, which preserve orminimize changes in the molecules in the sample. Such inhibitorsinclude, for example, chelators such as ethylenediamne tetraacetic acid(EDTA), ethylene glycol bis(P-aminoethyl ether) N,N,N1,N1-tetraaceticacid (EGTA), protease inhibitors such as phenylmethylsulfonyl fluoride(PMSF), aprotinin, leupeptin, antipain and the like, and phosphataseinhibitors such as phosphate, sodium fluoride, vanadate and the like.Appropriate buffers and conditions for isolating molecules are wellknown to those skilled in the art and can be varied depending, forexample, on the type of molecule in the sample to be characterized (see,for example, Ausubel et al., Current Protocols in Molecular Biology(Supplement 47), John Wiley & Sons, New York (1999); Harlow and Lane,Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory Press(1988); Harlow and Lane, Using Antibodies: A Laboratory Manual, ColdSpring Harbor Press (1999); Tietz Textbook of Clinical Chemistry, 3rded. Burtis and Ashwood, eds. W.B. Saunders, Philadelphia, (1999)). Asample also can be processed to eliminate or minimize the presence ofinterfering substances. For use in the methods described herein, asample can be in a variety of physical states. For example, a sample canbe a liquid or solid, can be dissolved or suspended in a liquid, can bein an emulsion or gel, and can be absorbed onto a material. As anon-limiting example, a sample can be a liquid blood sample, liquidserum sample, liquid white blood cell sample, dried blood, serum, orwhite cell sample, or such a sample absorbed onto a paper or polymersubstrate.

Exemplary Applications

The methods described herein can be used to obtain a molecular profilefor a sample, e.g., a sample from a subject such as a human. The profilecan include information that indicates whether succinylacetone, orsuccinylacetone and other biological analytes such as amino acids, ispresent and typically includes information about the presence (eitherqualitative or quantitative) of succinylacetone (and one or moreadditional biological analytes).

In some applications of these mass spectrometry methods, metabolicprofiles for a subject (e.g., a human) can be obtained. For example, theprofiles can include the level of succinylacetone in a subject (e.g., ahuman patient). Other biomolecules can also be detected, quantitated,and/or evaluated, including, e.g., one or more of an amino acid, freecarnitine, or an acylcarnitine, in a biological sample using tandem massspectrometry. The resultant information (metabolic profile) can be usedfor assessing the health state of a subject (e.g., a human patient),such as presence or absence of a metabolic disorder (e.g., an aminoacidopathy, a fatty acid or organic acid disorder, or a metabolicdisorder associated with altered levels of succinylacetone (e.g.,Hereditary tyrosinemia type I)), or for evaluating risk for such adisorder. Examples of amino acidopathies include, but are not limitedto, argininemia, argininosuccinic aciduria (argininosuccinate lyasedeficiency/argininosuccinase deficiency), citrullinemia(argininosuccinic acid synthetase deficiency/argininosuccinatesynthetase deficiency), homocystinuria, cystathione synthase deficiency,hypermethioninemia, hyperornithinemia, hyperammonemia,hyperhomocitrullinuria syndrome, ornithine translocase deficiency,hyperprolinemia, Maple Syrup Urine Disease (branched chainketoaciduria), nonketotic hyperglycinemia, phenylketonuria,pyroglutamic/pipecolic academia, tyrosenemia (Type I), tyrosenemia (TypeII), 5-oxoprolinuria, or pyroglutamic aciduria. Examples of fatty acidand organic acid disorders include, e.g., 2-methylbutyryl CoAdehydrogenase deficiency, 2,4-Dienoyl-CoA reductase deficiency,3-hydroxy-3-methylglutaryl CoA lyase deficiency (hydroxymethylglutaricacidemia), 3-methylcrotonyl CoA carboxylase deficiency(3-methylcrotonylglycinemia), carnitine palmitoyltransferase (type I)deficiency, carnitine palmitoyltransferase (type II) deficiency,carnitine transporter defect, carnitine/acylcarnitine translocasedefect, ethylmalonic academia, glutaric academia (type I; glutaryl CoAdehydrogenase deficiency); isobutyryl CoA dehydrogenase deficiency,Isovaleric academia, long-chain acyl-CoA dehydrogenase deficiency,long-chain hydroxyacyl-CoA dehydrogenase deficiency, malonic aciduria,medium-chain acyl-CoA dehydrogenase deficiency, methylmalonic academia,mitocondrial acetoacetyl CoA thiolase deficiency (Beta-Ketothiolasedeficiency), multiple acyl-CoA dehydrogenase deficiency (Glutaricacidemia, type II), multiple Co-A carboxylase deficiency(Holocarboxylase synthetase deficiency), propionic academia, short-chainacyl-CoA dehydrogenase deficiency, short-chain hydroxyacyl-CoAdhydrogenase deficiency, trifunctional protein deficiency, andvery-long-chain acyl-CoA dehydrogenase deficiency. Additional metabolicdisorders are described in, e.g., Chace et al. (2001) Clinical Chemistry47:1166-82; Rashed et al. (1997) Clinical Chemistry 43(7):1129-41;Schulze et al. (2003) Pediatrics 111(6):1399-1406; and Zytkovicz et al.(2001) Clinical Chemistry 47(11):1945-55.

Tyrosinemia type I (e.g., Hereditary tyrosinemia type I), for example,is caused by the lack of fumarylacetoacetase activity which leads to theaccumulation of fumarylacetoacetate (FIG. 1). Fumarylacetoacetate israpidly converted by other enzymes to succinylacetone and thus patientswith Tyrosinemia Type I accumulate succinylacetone in their blood.Therefore, the ability to detect succinylacetone either alone, ortogether with other biomolecules (e.g., metabolic biomolecules), can beuseful for assessing the health state of a subject. Hence, it ispossible to, at the same time, detect other amino acids such as tyrosineand methionine, as well and other biomolecules such as free carnitineand acylcarnitines, in the sample, e.g., by identifying unique peaks forsuch molecules in the mass spectrometry analysis. Table 1 includes anon-exhaustive list of analytes (e.g., biomolecules) that can bedetected/measured with succinylacetone (by way of derivatizedsuccinylacetone) using the methods described herein.

A metabolic profile obtained by the methods described herein can be usedin diagnosing or predicting susceptibility to a variety of metabolicdisorders because the biochemical indicators (e.g., succinylacetone)examined can be indicative of such disorders, whether or not physiologicor behavioral symptoms of the disorder have become apparent (e.g., onesuspected of having a metabolic disorder such as an amino acidopathy(e.g., tyrosinemia type I). A metabolic profile as described herein canbe useful for monitoring the metabolism of a subject (e.g., a mammalsuch as a human), such as one undergoing treatment for a metabolicdisorder. As a non-limiting example, the methods can be used fordetermining therapeutic efficacy of a particular treatment (e.g., theability of a treatment to restore levels of succinylacetone tophysiologic levels). Based on this determination, the subject can beoffered additional or alternative therapeutic options. The metabolicprofile can also be useful for assessing patient compliance with aparticular treatment modality, such as dietary restriction (e.g., theefficacy of a dietary regimen in restoring levels of succinylacetone tophysiologic levels). Therefore, the technology described herein isapplicable to screening, diagnosis, prognosis, monitoring therapy andcompliance, and any other application in which determining the presenceor amount of panels of two or more biomolecules, such as succinylacetoneand one or more of an amino acid, free carnitine, or an acylcarnitine,is useful.

A metabolic profile generated using the methods described herein can beobtained using a variety of biological samples. Suitable samples includethose described above.

In one aspect, a metabolic profile as described herein can be used toassess the presence or absence of a metabolic disorder such as an aminoacidopathy (e.g., tyrosinemia type I).

Subjects of all ages can be affected by metabolic disorders diagnosedusing a metabolic profile described herein. Therefore, a sample used ina method described herein can be obtained from a subject (e.g., a human)of any age, including a neonate, newborn, baby, child, and adult, suchas a pregnant female and individual having or suspected of havingtyrosinemia. The methods can also be used for individuals at risk ofdeveloping a metabolic disorder. Such individuals include those who have(i) a family history of (a genetic predisposition for) such disorders or(ii) one or more risk factors for developing such disorders. The methodscan also be used for prenatal diagnosis if the changes insuccinylacetone or at least one additional analyte (e.g., one or more ofan amino acid, free carnitine, or an acylcarnitine) levels are evidentin maternal samples such as amniotic fluid, maternal blood or plasma.The methods further can be used to monitor succinylacetone levels inindividuals having health conditions associated with alteredsuccinylacetone levels, such as individuals undergoing livertransplantation.

The methods described herein involve detecting the presence or amount ofsuccinylacetone and one or more additional biological analytes (e.g.,amino acids, free carnitine, or acylcarnitines, where the presence oramount of each biomolecule correlates the presence or absence of ametabolic disorder. The methods described herein can be usedquantitatively, if desired, to allow comparison of test sample resultswith known or a pre-determined standard amount of a particularanalyte(s) (e.g., by using an internal standard as described above). Themethods can also be used qualitatively when a test sample is comparedwith a reference sample, which can be either a normal reference ormetabolic disorder reference. In this format, the relative amount ofbiomolecules can be indicative of a metabolic disorder. A referencesample, for example, can be from a subject having, not suspected ofhaving, or not at risk of developing a disorder such as a metabolicdisorder such as an amino acidopathy (e.g., tyrosinemia type I).

Generally, a cut-off value for a given biomolecule can vary and would beknown in the art for commonly tested analytes and enzymes. Routine,obvious adaptations of methods known in the art can be used to establishcut-off values for uncommonly tested analytes. A cut-off value istypically a biomolecule amount, or ratio with another biomolecule, aboveor below which is considered indicative of a metabolic disorder or causefor retest. Thus, in accordance with the technology described herein areference level of at least one biomolecule in a particular sample typeis identified as a cut-off value, above which there is a significantcorrelation between the presence of the at least one biomolecule andpresence (or absence) of a metabolic disorder. It is understood thatbiomolecule panels can be interpreted as a whole, in parts or on ananalyte-by-analyte basis.

Those of skill in the art will recognize that some cut-off values arenot absolute in that clinical correlations are still significant over arange of values on either side of the cutoff; however, it is possible toselect an optimal cut-off value (e.g., varying H-scores, and the like)of biomolecule for a particular sample types. Cut-off values determinedfor use in the methods described herein generally are compared withpublished ranges but can be individualized to the methodology used andpatient population. It is understood that improvements in optimalcut-off values could be determined depending on the sophistication ofstatistical methods used and on the number and source of samples used todetermine reference level values for the different biomolecules andsample types. Therefore, established cut-off values can be adjusted upor down, on the basis of periodic re-evaluations or changes inmethodology or population distribution. In addition, instrument-specificcut-off values can be used, if desired, for example such as wheninter-instrument performance comparability is greater than 10%.

The reference level can be determined by a variety of methods, providedthat the resulting reference level accurately provides an amount of eachbiomolecule above which exists a first group of subjects (e.g., humans)having a different probability of metabolic disorder than that of asecond group of subjects having metabolic analyte or enzyme activityamount below the reference level. The reference level can be determinedby comparison of biomolecule amount in, e.g., populations of subjects(e.g., patients) having the same metabolic disorder. This can beaccomplished, for example, by histogram analysis, in which an entirecohort of patients are graphically presented, wherein a first axisrepresents the amount of biomolecule and a second axis represents thenumber of subjects in the cohort whose sample contain one or morebiomolecules at a given amount. Two or more separate groups of subjectscan be determined by identification of subsets populations of the cohortwhich have the same or similar levels of biomolecules. Determination ofthe reference level can then be made based on an amount which bestdistinguishes these separate groups. The reference level can be a singlenumber, equally applicable to every subject, or the reference level canvary, according to specific subpopulations of subjects. For example,older subjects can have a different reference level than youngersubjects for the same metabolic disorder. In addition, a subject withmore advanced disease (e.g., a more advanced form of a metabolicdisorder) can have a different reference value than one with a milderform of the disease.

The methods can also be used to determine the presence or amount ofother biologically active ketones, e.g., steroids. For example, themethods described herein can be used to detect the presence or amount ofa steroid in a biological sample obtained from a subject (e.g., a humanpatient), the methods can be used to diagnose, or generate metabolicprofiles useful for the diagnosis, of one or more conditions associatedwith altered levels of steroids, e.g., 21-OH deficiency, 11b-OHdeficiency, salt wasting 21-OH deficiency, adrenal cancer, adrenalhyperplasia, hypopituitarism, aldosterone synthase deficiency,adrenalcortical disorder, menopause, or pregnancy.

Methods of Identifying Compounds that Modulate Succinylacetone Levels

Also provided herein are methods of identifying compounds that modulate(e.g., decrease) the levels of succinylacetone in a cell. The compoundscan modulate succinylacetone and a number of additional biologicalmolecules such as, but not limited to, free carnitine, acylcarnitines,and amino acids (e.g., proline, methionine, or tyrosine). As discussedsupra, since disregulated (e.g., elevated) levels of succinylacetone areassociated with increased risk of certain disorders (e.g., aminoacidopathies), compounds so identified could be useful in treating aminoacidopathies such as tyrosinemia type I. Cells that can be contactedwith the candidate compound can be of any species such that the cellsproduce succinylacetone (either synthetically or naturally). The cellscan be primary cells or cell lines and can be of any histological type,e.g., without limitation, epithelial cells, fibroblasts, lymphoid cells,macrophages/monocytes, granulocytes, keratinocytes, neuronal cells, ormuscle cells. The cells can be cultured in tissue culture dishes. Oftenit is preferable to grow the cells in multiwell assay plates (e.g., 96well or 384 well assay plates) such that multiple candidate compoundscan be evaluated at one time. The candidate compound (optionally atvarious concentrations ranging, e.g., from 0.001 nM to 10 mM) can beadded to a solution (e.g., culture medium) containing the cells or,where the compound is a protein, the cells can recombinantly express it.Following incubation of cells expressing succinylacetone, the presenceor level of succinylacetone can be determined using the samplepreparation (extraction) and tandem mass spectrometry methods describedherein. Prior to detection, the cells can be lysed under conditions thatallow for a sample to be prepared, which is compatible with theextraction methods described herein and with tandem mass spectrometry.Often a control compound can be added to a set of cells as either apositive or negative control.

The compounds identified in any of the methods described herein includevarious chemical classes. Compounds can be biomolecules including, butnot limited to, peptides, polypeptides, peptidomimetics (e.g.,peptoids), amino acids, amino acid analogs, saccharides, fatty acids,steroids, purines, pyrimidines, derivatives or structural analoguesthereof, polynucleotides, and polynucleotide analogs. Compounds can beboth small and large molecule compounds.

Identification of test compounds through the use of the variouslibraries described herein permits subsequent modification of the testcompound “hit” or “lead” to optimize the capacity of the “hit” or “lead”to modulate the levels of succinylacetone in a cell.

The methods described herein can be modified to identify compounds thatmodulate the levels of a biologically active ketone, e.g., a steroid orany of the biologically active ketones described herein.

Kits

Also provided herein are kits useful for preparing samples for detectionand/or measurement (using tandem mass spectrometry) of succinylacetonealong with multiple other analytes (e.g., amino acids, free carnitine,and acylcarnitine) in a sample, e.g., a dried blood sample or any of thesamples described herein. The kits can be used to extractsuccinylacetone along with one or more additional analytes (e.g., aminoacids, acylcarnitines, and carnitines) from a sample (e.g., a bloodspot) in a single step such that the concentrations of succinylacetoneand additional analytes (e.g., amino acids, carnitine, andacylcarnitines) in the extract reflect their respective concentrations(or ratios) in the sample. The kits can be used to prepare a sample tosimultaneously screen succinylacetone, alanine, arginine, citrulline,glycine, leucine, methionine, ornithine, phenylalanine, proline,tyrosine, valine, and acylcarnitines such as CO, C2, C3, C3DC/C4OH, C4,C4DC/C5OH, C5, C5:1, C5DC/C6OH, C6, C6DC/C7OH, C8, C8:1, C10, C10:1,C10:2, C12, C12:1, C14, C14:1, C14:2, C14OH, C16, C16:1, C16OH, C16:10H,C18, C18:1, C18:2, C18OH, and C18:10 H (see Table 1).

The kits can include one or more internal standards and/or controls foruse in subsequent mass spectrometric analysis. For example, the kits caninclude succinylacetone (SA) as a control and a derivatized form oflabeled (e.g., isotope labeled) SA (e.g.,3,4,5,6,7-¹³C₅-(3-(5-methyl-1H-pyrazol-3-yl)propionic acid (MPP)) as aninternal standard. The succinylacetone and/or derivatizedsuccinylacetone can each be provided in the kit in a liquid or dried(e.g., lyophilized) form. The succinylactone and/or derivatizedsuccinylacetone can be provided in an amount of about 1 mmole (e.g.,about 1.5 mmole, about 2 mmole, about 2.5 mmole, about 3.0 mmole, about3.5 mmole, about 4.0 mmole, about 4.5 mmole, or about 5 mmole). The kitscan include succinylacetone or derivatized succinylacetone (e.g., MPP)in a container containing one or more additional controls or internalstandards. For example, the kit can include a container with asuccinylacetone control, one or more amino acid controls, and one ormore carnitine (e.g., free carnitine and acylcarnitines) controls. Thekits can also include proline as a control and stable, labeled (e.g.,isotope-labeled) proline as an internal standard.

The kits can also include a strong base such as hydrazine, e.g.,hydrazine dihydrochloride or any of the other strong bases describedherein. The base can be provided in solution at a concentration of lessthan about 0.5% (e.g., less an about 0.05%, less than about 0.06%, lessthan about 0.07%, less than about 0.08%, less than about 0.09%, lessthan about 0.1%, less than about 0.15%, less than about 0.2%, less thanabout 0.25%, less than about 0.3%, less than about 0.35%, less thanabout 0.4%, less than about 0.45%, or less than about 0.475%).

One or more solutions contained in the kit can be stored in, e.g.,silanized glass vials. One or more components of the kit can be storedin a container that prevents or minimizes loss of material orevaporation of a solvent. For example, the container can be sealed witha septum.

The kits can include, e.g., dried blood (e.g., plasma, lymph) spotsuseful as a control. For example, the dried blood spot can be enrichedwith one or more analytes (e.g., one or more analytes at knownconcentrations) such as succinylacetone, one or more amino acids, freecarnitine, or one or more acylcarnitines.

The kits can also, optionally, include an extraction solution such asany of the extraction solutions described herein. The extractionsolution can contain a C1-3 linear or branched monoalcohol and a strongbase. The kits can also include one or more solvent solutionscontaining, e.g., acetonitrile or isopropanol. The solvent solutions canalso contain water, e.g., a solvent solution containing 80% acetonitrileand 20% water.

In some embodiments, the kit can also include one or more components totest for biotinidase activity in a sample as well as to test for thepresence of lysosomal storage disorders or galactosemia in a subject.

Such kits can be used then in the detection of elevated or lowsuccinylacetone, amino acids, free carnitine, or acylcarnitine levels innewborn blood for the diagnosis of one or more of several metabolicdisorders. For example, elevated levels of succinylacetone can beindicative of tyrosinemia type I. Free carnitine and acylcarnitines aremarkers for disorders that are generally classified as fatty acidoxidation (FAO) disorders and organic aciduria disorders (OAD).Similarly, amino acids are used as markers for several metabolicdisorders collectively known as amino acidopathies. These disorders areinborn errors of metabolism (or genetic metabolic deficiencies).

It is understood that modifications that do not substantially affect theactivity of the various embodiments of this invention are also includedwithin the definition of the invention provided herein. Accordingly, thefollowing example is intended to illustrate, not to limit, the presentinvention.

EXAMPLES Example 1

Reference standard blood (whole blood) spots were prepared using wholeblood obtained from a subject. The blood was processed by adjusting thehemoglobin concentration to 17 mg/dL and adding to the blood severalamino acids, carnitine, acylcarnitines and succinylacetone at knownconcentrations. The processed blood was dispensed onto filter papercards to form blood spots on the filter paper matrix. Each blood spotwas generated by dispensing 75 μL of processed blood. The blood spotswere allowed to dry overnight.

A small disc (⅛″) of a dried blood spot was punched and deposited in awell of a micro titer plate well. The sample was extracted by dispensing100 μL of an extraction solution that consisted of a mixture of methanoland water at an approximate relative volume-to-volume ratio of 78%methanol and 22% water. In addition, the extraction solution contained a3 mM oxalic acid, and a concentration of 600 μM of hydrazinedihydrochloride. Internal standards (stable heavy isotope analogs of theanalytes of interest) for several amino acids, carnitine,acylcarnitines, and succinylacetone (MPP) were also present in theextraction solution. The internal standards included in the solution areindicated in tandem mass spectrometry scan shown in FIG. 5.

The extracted sample was injected into an electrospray triple quadrupoletandem mass spectrometer with the aid of an automated liquid handlingdevice. Mass spectral data for the amino acids and succinylacetone (MPP)were acquired via a neutral loss of 46 scan (FIG. 5).

Example 2

Blood spots containing several amino acids, carnitine, acylcarnitinesand succinylacetone at known concentrations were prepared as above(Example 1). A small disc (⅛″) of a dried blood spot was punched anddeposited in a well of a micro titer plate well. The sample wasextracted in the presence of internal standards as described above.

Following extraction, the sample was evaporated to dryness. The driedsample was then reconstituted in 3N HCl in n-butanol and incubated at39° C. for about 30 minutes. Following this incubation, the sample wasagain evaporated to dryness and then reconstituted in a solution ofacetonitrile and water.

The extracted sample was injected into an electrospray triple quadrupoletandem mass spectrometer with the aid of an automated liquid handlingdevice. The data was acquired in the neutral loss of 102 scan. Theformation of butyl esters is evident by the 56 dalton (Da) increase(cross reference FIG. 5) in the m/z of the ions brought about by thisesterification (FIG. 6). These data demonstrate that following thederivatization of succinylacetone (as described herein), the sample canbe further processed (e.g., by esterification), if need be, to detectother analyte constituents.

Example 3

Dried blood spots were prepared as above. The blood spots were spikedwith different levels of the analytes (amino acids, Succinylacetone(SA), free carnitine and acylcarnitines) shown in Table 2 and Table 3.The blood spots were extracted as described above (Example 1; thedefinition for each of the analytes indicated in the tables can be foundin Table 1.) The extracted sample was injected into an electrospraytriple quadrupole tandem mass spectrometer with the aid of an automatedliquid handling device. The mass spectral data for the amino acid andsuccinylacetone (MPP) was acquired via a neutral loss of m/z 46 scan andfor carnitine and acylcarnitines via a precursor ion of m/z 85 scan. Thepercentage of each analyte recovered was determined through comparisonwith an internal standard for each analyte.

The various recoveries, by percentage, are presented in Table 2 thevarious levels of precision are presented in Table 3.

The imprecision of the assay was determined by analyzing the samplesdescribed in table three. Each sample run consisted of triplicatepunches of each sample which were processed and measured as described inExample 1. The study included two such runs a day for a total of fivedays. With this information the following imprecision components weredetermined: within run, between run—within day, and between day fromwhich the total imprecision was determined. The results of theimprecision analysis are shown in Table 3.

These data demonstrate that the methods described herein can be used tosimultaneously extract and quantify MPP, amino acids, carnitine andacylcarnitines using tandem mass spectrometry.

Example 4

Tyrosine is currently used as a diagnostic marker for screeningtyrosinemia type I. To show that detection of succinylacetone, ascompared to tyrosine, results in an increase in specificity fordetermining tyrosinemia type I status in an individual, blood samplesfrom an affected patient (i.e., tyrosinemia type I positive patient)were compared to known normal samples for the corresponding tyrosine andsuccinylacetone (MPP) concentrations. Dried blood spots from healthynewborns and from a newborn with a confirmed case of tyrosinemia type Iwere obtained at 25 hours and 14 days of age. The blood spots wereextracted and subjected to mass spectrometric analysis as describedabove (Example 1). Although the affected patient displays normaltyrosine levels at 25 hours of age (the newborn screening window), it isnot until the patient is 14 days old that the tyrosine levels aresignificantly elevated (FIG. 7 spectra and FIG. 8; table and bargraphs). In contrast, succinylacetone (MPP) shows very significantelevation (30-40 standard deviations form the normal mean) even as earlyas 25 hours after birth. Thus, at 25 hours of age, this patient wouldhave been a false negative if tyrosine would have been the only markerused. Since early detection is crucial for tyrosinemia type I, detectingsuccinylacetone is very advantageous for diagnosing this condition.

Example 6

Many of the methods described herein use a strong base to form a Schiffbase with succinylacetone so it can be extracted and measured. Hydrazinecan be obtained in several forms, e.g., hydrazine hydrate or hydrazinedihydrochloride. Although both of these forms perform similarly in themethods described herein, to test which of the two forms of hydrazineare the most stable, and thus would have the longest shelf-life, therelative stability of each form was tested over a time span about 60days. Solutions of each form of hydrazine (hydrazine hydrate at 0.5% andhydrazine dihydrochloride at 0.1%) were incubated at 30° C. and atvarious time points (e.g., 1 to about 60 days), the amount of eachhydrazine form was determined by a standardized flruorometric assay.Hydrazine hydrate was determined to be unstable, whereas, hydrazinedihydrochloride was determined to be much more stable and thus much moresuitable for a robust product (FIG. 9).

TABLE 2 Percent recoveries for various analytes in dried blood. ALA ARGCIT GLY LEU MET SA ORN PHE PRO TYR VAL Spike (μM) 81 79 31 83 45 21 4 6842 63 54 41 Recovery 67 70 90 83 68 74 72 100 101 96 94 78 Spike (μM)644 635 251 665 357 171 29 544 337 507 431 324 Recovery 77 71 86 79 7073 69 94 99 94 93 83 Spike (μM) 1450 1429 564 1495 803 385 65 1223 7591141 969 730 Recovery 78 71 83 78 70 72 66 92 99 92 91 83 Spike (μM)3261 3216 1270 3364 1807 866 147 2752 1708 2567 2181 1642 Recovery 90 8092 88 79 81 76 101 109 104 102 93 C0 C2 C3 C4 C5 C5DC C6 C8 C10 C12 C14C16 C18 Spike (μM) 45 14 1.7 1.3 1.2 0.5 1.2 0.8 0.5 0.9 0.8 1.5 0.5Recovery 101 79 72 71 80 102 83 80 85 82 87 84 90 Spike (μM) 362 110 1410 10 4 10 7 4 7 6 12 4 Recovery 94 77 73 68 76 97 80 76 80 78 84 82 79Spike (μM) 814 248 31 24 21 9 21 15 9 15 14 28 10 Recovery 92 75 71 6674 95 77 73 78 75 83 81 80 Spike (μM) 1831 557 70 53 48 21 48 33 20 3531 62 22 Recovery 102 83 81 75 82 104 87 80 88 85 92 90 88${{Percent}\mspace{14mu} {recovery}} = {\frac{\left( {{measured}\mspace{14mu} {concentration}} \right) - \left( {{endogenous}\mspace{14mu} {concentration}} \right)}{{Spiked}\mspace{14mu} {concentration}} \times 100}$

TABLE 3 Total imprecision. ALA ARG CIT GLY LEU MET SA ORN PHE PRO TYRVAL C0 Measured μM 567.1 30.0 29.1 327.7 185.6 27.2 0.8 127.2 84.7 275.583.8 206.2 51.0 % CV 8 7 11 10 7 7 25 7 7 9 8 8 7 Measured μM 604.1 51.139.7 359.0 201.2 33.5 1.7 152.5 102.2 304.4 104.1 222.9 67.9 % CV 9 7 109 7 8 21 7 8 9 7 8 7 Measured μM 709.9 135.6 81.6 463.1 251.8 57.0 5.4254.6 166.3 398.9 182.0 278.6 134.9 % CV 8 6 8 8 6 7 12 6 7 6 7 7 8Measured μM 1104.2 476.8 243.9 867.0 447.1 152.3 20.5 637.2 419.5 763.4482.8 489.2 390.0 % CV 7 7 9 9 6 7 10 7 8 9 7 8 6 C2 C3 C4 C5 C5DC C6 C8C10 C12 C14 C16 C18 Measured μM 36.4 3.3 0.3 0.3 0.2 0.1 0.1 0.1 0.1 0.22.2 2.1 % CV 7 9 11 11 13 12 13 12 10 10 8 7 Measured μM 42.0 4.0 0.70.6 0.4 0.5 0.4 0.3 0.4 0.4 2.7 2.3 % CV 8 11 10 10 12 10 9 10 8 8 7 6Measured μM 60.8 6.3 2.2 2.0 1.2 2.0 1.3 0.9 1.5 1.4 4.6 2.9 % CV 7 8 87 10 8 8 9 7 7 6 6 Measured μM 134.6 15.3 8.3 7.4 4.2 7.7 5.0 3.3 6.05.3 12.2 5.5 % CV 7 9 10 7 8 8 7 7 7 7 8 10 Total % CV includes: withinrun, between run -within day, and between day imprecision.

Other embodiments are within the scope of the following claims.

1. A method for detecting succinylacetone, the method comprising:contacting a sample with an extraction solution comprising methanol anda strong base; derivatizing succinylacetone in the sample; andevaluating the derivatized succinylacetone in the derivatized sampleusing tandem mass spectrometry.
 2. A method for detectingsuccinylacetone, the method comprising: contacting a sample with anextraction solution comprising methanol and hydrazine; derivatizingsuccinylacetone to 3-(5-methyl-1H-pyrazol-3-yl) propionic acid (MPP) inthe sample; and evaluating MPP in the derivatized sample using tandemmass spectrometry.
 3. The method of claim 2, further comprisingevaluating the sample for one or more additional analytes.
 4. The methodof claim 3, wherein the one or more additional analytes are evaluatedwith MPP in the same sample injection.
 5. A method for detectingsuccinylacetone, the method comprising: contacting a sample with anextraction solution containing an organic solvent under conditions thatdo not substantially fix proteins; derivatizing succinylacetone to3-(5-methyl-1H-pyrazol-3-yl) propionic acid (MPP) in the sample; andevaluating MPP and an additional analyte (or derivative thereof) in thederivatized sample using tandem mass spectrometry.
 6. The method ofclaim 2, wherein the extraction solution comprises at least about 5%water.
 7. The method of claim 2, wherein the extraction solutioncomprises less than about 85% methanol.
 8. The method of claim 2,wherein the succinylacetone is derivatized with hydrazine or derivatizedhydrazine.
 9. The method of claim 2, wherein the sample is a dried bloodsample.
 10. The method of claim 2, wherein the sample is from a newbornhuman.
 11. The method of claim 2, further comprising determining whethera subject, from whom the sample was derived, has, or is at risk ofdeveloping, hereditary tyrosinemia type I, based on the detection ofsuccinylacetone in the sample.
 12. The method of claim 11, furthercomprising after determining that the subject has, or is at risk ofdeveloping, hereditary tyrosinemia type I, administering to the subjectan inhibitor of 4-hydroxyphenylpyruvate dioxygenase.
 13. The method ofclaim 2, wherein a derivatized succinylacetone comprising at least oneheavy atom isotope is included in the sample prior to mass spectroscopicanalysis.
 14. A method for detecting a biologically active ketone, themethod comprising: contacting a sample with an extraction solutioncomprising methanol and a strong base; derivatizing a biologicallyactive ketone in the sample; and evaluating the derivatized biologicallyactive ketone in the derivatized sample using tandem mass spectrometry.15. The method of claim 14, wherein the biologically active ketone issuccinylacetone.
 16. The method of claim 14, wherein the biologicallyactive ketone is a steroid.
 17. A kit for detecting succinylacetone, thekit comprising: derivatized succinylacetone comprising at least oneheavy atom isotope; and instructions for how to detect the derivatizedsuccinylacetone.
 18. The kit of claim 17, wherein the derivatizedsuccinylacetone is 3-(5-methyl-1H-pyrazol-3-yl) propionic acid (MPP).19. The kit of claim 17, further comprising a strong base.
 20. The kitof claim 19, wherein the strong base is hydrazine.
 21. The kit of claim20, wherein the hydrazine is hydrazine dihydrochloride.
 22. The kit ofclaim 20, wherein the hydrazine is in solution at a concentration ofless than about 0.1%.
 23. The kit of claim 17, further comprising one ormore internal standards, each internal standard comprising: (i) an aminoacid, free carnitine, or an acylcarnitine and (i) at least one heavyatom isotope.
 24. The kit of claim 17, further comprising at least onedried blood spot comprising a known amount of one or more ofsuccinylacetone, an amino acid, free carnitine, or an acylcarnitine. 25.A method for extraction, the method comprising: contacting a sample withan extraction solution, the extraction solution comprising methanol anda strong base, wherein contacting the sample with the extractionsolution yields an extract comprising (i) derivatized succinylacetone,(ii) one or more amino acids, (iii) free carnitine, (iv) one or moreacylcarnitines or (v) a derivatized form of (ii), (iii), or (iv) fromthe sample, wherein the concentration of the derivatized succinylacetonein the extract reflects the concentration of succinylacetone in thesample, and wherein the concentrations of the one or more amino acids,free carnitine, one or more acylcarnitines, or derivatized forms thereofin the extract reflect their respective concentrations in the sample.26. The method of claim 25, further comprising after contacting thesample with the extraction solution, analyzing the sample using tandemmass spectrometry.
 27. The method of claim 25, wherein the contactingderivatizes at least one succinylacetone molecule to3-(5-methyl-1H-pyrazol-3-yl) propionic acid (MPP).
 28. The method ofclaim 25, wherein the sample is a biological sample.
 29. The method ofclaim 28, wherein the biological sample is a dried blood sample.
 30. Themethod of claim 25, wherein the extraction solution further compriseswater.
 31. The method of claim 25, wherein the extraction solutioncomprises between about 5% to about 30% water.
 32. The method of claim25, wherein the strong base is hydrazine.
 33. The method of claim 25,wherein the strong base is a modified hydrazine.
 34. The method of claim25, wherein the extraction solution further comprises an organic acid.35. The method of claim 34, wherein the organic acid is oxalic acid. 36.The method of claim 25, wherein the extraction solution furthercomprises one or more internal standards.
 37. The method of claim 25,further comprising after contacting the sample with the extractionsolution, evaporating the sample resulting in a first evaporated sample.38. The method of claim 37, further comprising after evaporating thesample, contacting the first evaporated sample with an alkyl alcoholsolution comprising an alkyl alcohol and an acid.
 39. The method ofclaim 38, wherein the alkyl alcohol is n-butanol.
 40. The method ofclaim 38, wherein the acid is HCl.
 41. The method of claim 38, furthercomprising after contacting the sample with the alkyl alcohol solution,evaporating the solution resulting in a second evaporated sample. 42.The method of claim 41, further comprising reconstituting the secondevaporated sample.
 43. The method of claim 42, wherein reconstitutingcomprises contacting the second evaporated sample with a solventcomprising acetonitrile.